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Lab 06 P/O Ratios

Introduction to P/O Ratios and Control Ratios

P/O and control ratios indicate the degree of coupling between electron transport and biochemical production of ATP. As such, they are a key measure of organelle integrity, and are routinely used to assess physiological status of not just chloroplasts, but mitochondria as well. The lab exercises on P/O Ratios and Carbon Dioxide Coupling are a test of your Experimental Hands. That is, how good you are at isolating high quality chloroplast suspensions that are as intact as possible.

The basic principle is to determine the rate of electron transport (measured by O2 production) with Pi and Mg present, but no ADP; then add a small, known amount of ADP. During phosphorylation (Pi + ADP –––> ATP), the rate of electron transport should be measurably faster (resulting in a faster rate of O2 production). As ADP is consumed, the rate of electron transport will decline, possibly to a rate slower than before ADP addition, since the product of phosphorylation, ATP, is now present.

The control ratio (a ‘respiratory control ratio’ for mitochondria, a ‘photosynthetic control ratio’ for chloroplasts) is the ratio of the rates of electron transport after/before adding ADP. Since ATP is present during the largest part of the time that ADP is present, normally, the best control is the electron transport rate without ADP, but with ATP present.

Since electron transport is faster during phosphorylation, it is possible to estimate the amount of electron transport required to phosphorylate a known amount of ADP. This is known as the P/O ratio. Please note that this is only an indirect estimate and cannot be considered a substitute for a direct measurement of the ATP produced by a measured amount of electron transport. Even so, it is commonly used to assess coupling, and therefore intactness of the organelle.

Isolation of Intact Chloroplasts

To assure that the chloroplasts are isolated as intact as possible, the media used during isolation are more elaborate than those used to measure fluorescence of the thylakoid membranes.

Solutions

Grinding Buffer (pre-chilled on ice)
Sucrose (FW 342.3)0.4 M
Choline-Cl (FW 139.6)0.2 M
HEPES (FW 238.3)20 mM(pH to 7.8 at 0º C with KOH)
MgCl2•6H2O (FW 203.3)5 mM
Sodium ascorbate (FW 216.1)2 mM(a reductant that protects the chloroplasts from oxidation)
BSA (bovine serum albumen, fraction V)2 mg/ml
Resuspension Buffer (pre-chilled on ice). The same as the grinding buffer, but without sodium ascorbate or BSA.


Other materials:

  • Spinach
  • Mortar and pestle (pre-chilled on ice)
  • Cheesecloth
  • Centrifuge and centrifuge tubes
  • N2 gas cylinder (to bubble through assay solutions to remove O2)
  • Miscellany (a small artist’s brush, beakers, cuvettes, No. 1 filter paper etc.)

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Chloroplast Isolation Protocol

After pre-washing the leaves and removing midrib veins, grind about 25 grams of the leaves in a pre-chilled mortar and pestle in 150 ml of the grinding medium.

Be sure to rinse the leaves thoroughly! At least 5 times! Bacteria growing on the leaves can ‘co-purify’ with the chloroplasts, resulting in O2 consumption as a consequence of bacteria respiring (another use of the Clark electrode, in microbiological diagnostics). Ensuring the media are ice-cold minimizes bacterial growth.

The mortar and pestle is a gentler homogenizing method than the blender; it leaves the chloroplasts more intact, but will result in a lower yield of chloroplasts. The homogenate is strained through three layers of cheesecloth into a pre-chilled beaker in ice. Centrifuge for 1–2 min at 500 X g to spin down debris. The supernatant should be centrifuged for 10 min at 10,000 X g to spin down the chloroplast into a pellet. Decant the supernatant and gently disperse the pellet into about 5 ml of the resuspension buffer using the fine brush. Keep the chloroplast suspension on ice.

Check the chlorophyll concentration and adjust to 0.5 mg/ml. Use 0.50 ml (250 micrograms chlorophyll) per 2 ml of reaction mix in the oxygen electrode measurements.

Measuring P/O and Control Ratios

Solutions:

Standard Buffer (bubble with N2 to remove ambient levels of O2)
Tricine (FW 179.2)100 mM(pH 8.2 with NaOH)
Sorbitol (FW 182.2)50 mM
NaCl (FW 58.44)100 mM
BSA (bovine serum albumen, fraction V2 mg/ml



Stock solutions

  • K-ferricyanide 100 mM
  • sodium phosphate (pH 8.2) 100 mM
  • MgCl2•6H2O 100 mM
  • NH4Cl 300 mM
  • ADP and ATP 6 mM (each)(keep on ice)

Mix in a test tube:

  • 1.0 ml of standard buffer
  • 0.5 ml of chloroplasts
  • 0.1 ml of sodium phosphate
  • 0.1 ml of MgCl2•6H2O
  • 0.1 ml of K-ferricyanide
  • 0.1 ml of dH2O

You may need to de-gas by N2 bubbling the reaction mix if there is too much oxygen – the best results will be obtained if O2 levels are low at the start of the experiment.

Load the oxygen electrode cell with this mixture, insert the stopper, then let the cell come to an equilibrium temperature. Illuminate with a bright light until oxygen evolution has achieved a steady rate. Then inject 0.05 ml of ADP into the oxygen electrode cell. Observe the rate of oxygen evolution. Once the faster burst of oxygen evolution is complete and the rate has slowed to a steady rate, add an additional 0.05 ml of ADP.

Increasing the gain and adjusting the offset should give you a clearer recording of the change in oxygen levels. Be sure to annotate what you do! A change in the gain must be accounted for in your calculations.

In a second run, add 0.05 ml of ATP first, then add 0.05 ml of ADP. After oxygen evolution has returned to its slower rate, add 0.2 ml of NH4Cl.

NH4Cl is an uncoupler, which ‘quenches the ∆H+ gradient across the thylakoid membrane by consuming H+ so that ATP synthesis is no longer coupled to the electron transport chain.

Calculations

  1. Rates of electron transport as nmole O / (mg Chl) / hour for all reactions
  2. The control ratio: Rate(+ADP) / Rate(+ATP)
  3. Uncoupled ratio: Rate(+NH4) / Rate(+ATP)
  4. Amount of O2 consumed as nmoles O during the faster (phosphorylation) rate of electron transport
  5. P/O ratio as the amount ADP added / amount of O2 consumed (part (4) above).

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